Certain nucleosides are currently employed as antiviral agents, or as anticancer agents and it is known that they must be metabolized in their triphosphorylated form in order to exert an antiviral or anticancer inhibitory activity.
In the context of antiviral treatments, notably in anti-HIV therapies, it is important to quantify these triphosphorylated forms of the nucleoside analogues.
Thus, with the goal of quantifying the active form of the nucleoside analogues, it is essential to use sensitive quantitative determination methods that are specific to said triphosphorylated nucleosides, referred to below as “ddNTPs.”
One of the determinant factors of the therapeutic failure of anti-HIV treatments can be explained by an alteration of the cellular metabolism for nucleosides (ddNs), which leads to an insufficient concentration of the active molecule in the intracellular medium. Therefore, one of the means of controlling loss of therapeutic efficacy would be the determination of the intracellular concentration of these molecules (triphosphorylated nucleosides and antiproteases) by immunological determinations. The therapeutic drug monitoring (TDM) of patients can then be envisaged for optimizing the administration of these molecules by proposing personalized treatments, but also being able to evaluate drug combinations.
The very intense catabolism of the antiproteases by the hepatic and intestinal enzymes, as well as the mechanisms of excretion, result in the administration of extremely high doses of antiviral agents to patients which can lead to devastating side effects.
The problem is different with regard to the nucleoside analogues. The doses administered are very high because of the limited penetration of the precursor into cells. The different phosphorylated metabolites of the drug will thus affect not only the viral polymerases, but also the cellular polymerases. The toxicity of the catabolites of the drug will be added to the nonspecificity of the nucleoside analogues. In anti-HIV treatment, because of the high quantities of antiviral agents administered to the patients, drug catabolites would affect a large number of cellular compartments including those which are reserved for the differentiation of the stem cells, thereby inducing the side effects which make anti-HIV treatments so constraining and uncomfortable.
Thus, the plasma and intracellular levels of those anti-HIV molecules which are used most often in combination directly reflect the metabolism of the entire organism. Thorough knowledge of the plasma and intracellular levels of these anti-HIV molecules is essential, and differs from individual to individual and even over time in the same patient.
Measurement of these parameters would enable clinicians to determine the consequences of combination regimes, and to optimize the use of anti-HIV drugs and to adjust the dosage as a function of the patient and the evolution of his disease. It is therefore important for the clinician to have access to the measurement of the intracellular content, which has a very close relationship with the plasma concentration and viral load. This viral load has a direct impact on the therapeutic success.
Methods of intracellular quantitative determinations of nucleoside derivatives have been developed; See, e.g., Robbins, B. L. et al. (1996) “Quantification of intracellular zidovudine phosphates by use of combined cartridge-radioimmunoassay methodology”. Antimicrob. Agents Chemother. 40, 2651-2654; Goujon, L. et al. (1998) “Monitoring of intracellular levels of 5′-monophosphate-AZT using an enzyme immunoassay”. J. Immunol. Methods, 218, 19-30; Robbins, B. L. et al. (1998) A. “Development of a new cartridge radioimmunoassay for determination of intracellular levels of lamivudine triphosphate in the peripheral blood mononuclear cells of human immunodeficiency virus infected patients”. Antimicrob. Agents Chemother. 42, 2656-2660).
However, these methods do not have adequate test sensitivity, which is especially required for detecting the low concentrations of nucleoside analogues present in the cell compartments and available in small samples.
Although methods exist for the measurement of nucleosides using HPLC and CE (capillary electrophoresis), the exciting methods are difficult to apply for the therapeutic monitoring of patients. These existing methods require a meticulous preparation of the samples, involve a lengthy analysis time and need a large blood volume to enable detection of the low intracellular levels. Furthermore, the investment costs required for purchasing the equipment and reagents for its operation, such as solvents and columns, represents a major drawback for laboratories and hospitals.
With regard to the phosphorylated derivatives of nucleoside analogues, there does not exist a method of chromatographic determination. In contrast, immunologic methods exist but they are indirect and require multiple steps, among others the purification of the phosphorylated metabolites, enzymatic dephosphorylation or elimination of the salts and reagents. (Robbins, B. L. et al. (1996) “Quantification of intracellular zidovudine phosphates by use of combined cartridge-radioimmunoassay methodology”. Antimicrob. Agents Chemother. 40, 2651-2654; Goujon, L. et al. (1998) “Monitoring of intracellular levels of 5′-monophosphate-AZT using an enzyme immunoassay”. J. Immunol. Methods, 218, 19-30; Robbins, B. L. et al. (1998) A. “Development of a new cartridge radioimmunoassay for determination of intracellular levels of lamivudine triphosphate in the peripheral blood mononuclear cells of human immunodeficiency virus infected patients”. Antimicrob. Agents Chemother. 42, 2656-2660).
Moreover, certain methods have been envisaged for obtaining such antibodies, e.g., antibodies directed against AZT-TP, using as immunogen an AZT-TP derivative whose phosphoric anhydride bonds have been substituted by methylene phosphonate bonds (or P—CH2—P). The methylene-bis-phosphonate group (P—CH2—P—CH2—P) possesses structural characteristics close to those of the pyrophosphate group (P—O—P—O—P), since the angle of the phosphoric anhydride bond (P—O—P) is 130°, that of the methylene phosphonate bond is 117°; the length of the P—O bonds is 1.61 Å and that of the P—C bonds is 1.79 Å. (Saady et al. (1995), “First synthesis of fully deprotected diimidotriphosphoric acid and derivatives designed for the synthesis of “PNPNP” nucleotides and dinucleotides”. J. Org. Chem. Vol. 60, pp 3685-3691).
However, the affinity of the different antibodies directed against this methylene-bis-phosphonate analogue of AZT-TP is not sufficient to enable its quantification, especially since the intracellular quantities of nucleotides are extremely small and the sample volume is also reduced.